Two forms of vitamin D exist in nature: vitamin D2 (ergocalciferol), which is formed in plants by the UV irradiation of the plant product ergosterol, and vitamin D3 (cholecalciferol), which is formed in animal tissues from near-UV (290-310 nm) irradiation of 7-dehydrocholesterol found in keratinocytes (123). In animals, vitamin D3 functions as a key regulator of mineral ion homeostasis, but first the vitamin must undergo two modifications in order to be activated. In the liver, vitamin D3 is initially hydroxylated at position 25, and in the kidney, it is subsequently hydroxylated at position 1 to produce 1,25-(OH)2D3, the hormonal form of vitamin D (1). Upon reaching its target tissues, 1,25-(OH)2D3 binds to its specific nuclear receptor, the vitamin D receptor (VDR), to regulate the transcription of vitamin D target genes responsible for carrying out physiological actions including: mineral homeostasis, skeletal homeostasis, and cellular differentiation (2).
The Cyp24a1 gene encodes the CYP24A1 cytochrome P450 enzyme that catalyzes the addition of a hydroxyl group on carbon 24 of the vitamin D secosteroid backbone. When the substrate is 1,25-(OH)2D3, hydroxylation by CYP24A1 leads to the production of 1,24,25-trihydroxyvitamin D3. 1,24,25-trihydroxyvitamin D3 is the initial reactant in the 24-oxidation pathway that leads to metabolite inactivation (3). Indeed, blocking CYP24A1 cytochrome P450 activity in cell culture systems inhibits catabolism of, and results in increased accumulation of 1,25-(OH)2D3 (4). The function of the CYP24A1 protein as an effector of 1,25-(OH)2D3 breakdown has also been confirmed in vivo. For example, mice deficient for the Cyp24a1 gene cannot effectively clear 1,25-(OH)2D3 from their circulation (5).
The 25-(OH)D3 metabolite can also serve as the substrate for the CYP24A1 enzyme. Use of 25-(OH)D3, as the substrate leads to the production of 24,25-(OH)2D3. Prior to the filing of this application, the potential bioactivity of 24,25-(OH)2D3 remained controversial. For example, the literature demonstrates that Cyp24a1 is expressed in growth plate chondrocytes and that cells from the growth plate respond to 24,25-(OH)2D3 in a cell maturation-dependent manner (6). However, the growth plates from Cyp24a1−/− mice do not show major defects (5). These observations suggested that the absence of CYP24A1 activity does not affect growth plate development and that 24,25-(OH)2D3 is not required for chondrocyte maturation in vivo.
Another aspect of bone biology in which investigators have sought to identify a role for 24,25-(OH)2D3 is fracture repair. Traumatic injury is a major public health issue. In the United States, close to 10 million trauma-induced fractures are reported annually (National Center for Health Statistics). United States statistics for the year 2002 reported 54 million office visits, 21 million emergency room visits, 4.5 million outpatient visits and 2 million hospitalizations dealing with traumatic injuries. Of the 2 million hospitalizations, 1.3 million related to bone fractures (United States Bone and Joint Decade web site, www.usbjd.org). Fractures continue to be the leading cause of injury hospitalization in the United States, accounting for more than one-half of all injury hospitalizations in 2004-2005 (National Center for Health Statistics).
With these traumatic fracture statistics in mind, consideration must also be given to the increase in the incidence of osteoporotic fractures that occurs in individuals after age 65. The aging of the U.S. population will increase the relative impact of musculoskeletal conditions: over the next thirty years, the percent of the population age 65 and over will increase from 12.8% to 20.0%. Individuals 65 years and older, especially women, are more likely to sustain a bone fracture. Each year, roughly 1.5 million people suffer a bone fracture related to osteoporosis (FDA Consumer magazine, January-February 2005 issue).
It has previously been shown that circulating levels of 24,25-(OH)2D3 increase during fracture repair in chicks due to an increase in renal CYP24A1 activity (7). When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25-(OH)2D3 alone resulted in poor healing (8). However, the strength of healed bones in chickens fed 24,25-(OH)2D3 in combination with 1,25-(OH)2D3 was equivalent to that measured in a control population fed 25-hydroxyvitamin D3 (8). Such results support a role of 24,25-(OH)2D3 as an essential vitamin D metabolite for fracture repair in chickens. Furthermore, in light of the signaling pathway associated with 1,25-(OH)2D3 in chickens, it was postulated that 24,25-(OH)2D3 also acts through receptor-mediated signaling, and preliminary evidence suggested the presence of a non-nuclear membrane receptor for 24,25-(OH)2D3 in the chick tibial fracture-healing callus (9,10). Prior to the instant application, studies establishing a therapeutic activity for 24,25-(OH)2D3 in mammalian fracture repair and the molecular nature of a 24-hydroxylated vitamin D compound receptor had not been reported.
At present, the only drugs approved for fracture repair/treatment are recombinant Bone Morphogenetic Proteins (BMPs). Their use is restricted to anterior lumbar interbody spine fusion, open tibial shaft fractures, and recalcitrant nonunion fractures (14). These treatments are extremely costly and success rates remain below 70% (15). The pharmaceutical industry is working on smaller and cheaper molecules that could activate the BMP receptors (BMP mimetics), however there have been no published results on such studies.
Although not yet approved for human use, parathyroid hormone (“PTH”) administration has been shown to improve fracture repair in rat studies (16, 17). However, the most dramatic effects come relatively late during the repair process. Furthermore, while parathyroid hormone treatment has few side effects, it is costly and requires daily injections (18). Accordingly, it is not likely to be a treatment that will be well tolerated by many patients.
Selective prostaglandin receptor agonists have also been considered for stimulation of bone repair. For example, selective agonists for receptor E2 (EP2) and receptor E4 (EP4) have been shown to stimulate fracture repair in rodents and dogs (19, 20). However, whether these agonists are being further developed for clinical use is unknown, as is the potential that significant undesired side effects may be associated with their use.
It has also recently been shown that bone mass can be regulated from the hypothalamus via the nervous system through adrenergic receptors (25). Based on that finding, it was hypothesized that bone mass may be susceptible to modulation by compounds that block such receptors (“beta-blockers”). For example, propranolol, a common beta-blocker, was shown to increase bone mass in wild-type mice and repair bone defects in rats (25, 27). Indeed, a large case-control study has suggested that beta-blockers reduce the risk of osteoporosis fractures (26). However, whether beta-blockers can be used to improve bone fracture healing without eliciting significant side effects has not been determined.
Finally, lipid-lowering drugs, known as statins, have also been shown to stimulate bone formation in vitro and in rodents (21). For example, there has been a report of enhanced fracture repair in mice following simvastatin treatment (22). However, the effectiveness of statin treatment for osteoporosis and fracture repair treatment remains controversial. The lack of association in randomized trials and the heterogeneity among observational studies do not support an effect of statins in preventing fractures (23). This could be due to the pharmacokinetic properties of statins, which are rapidly metabolized after one passage through the liver (24). Thus, it remains questionable whether statins could be used efficaciously for treatment of bone injuries.
Specifically, there is a need for a therapy that is less costly and more easily administerable than the therapies discussed above and that has an acceptable side effect profile.
The instant invention addresses the deficiencies of the compounds currently under study by providing new avenues for identifying compounds having more desirable traits. Specifically, the instant invention relates to novel 24-hydroxylated vitamin D compound receptor which can be employed in the development of such compounds. In addition, the instant application provides the first data establishing that 24-hydroxylated vitamin D compounds can function as a therapeutic in mammalian fracture repair.